An essential step in gene expression in eukaryotic cells is the processing of pre-mRNA into functional mRNA for protein synthesis. The fundamental question here is how intronic sequences are efficiently and precisely removed. Splicing defects due to mutations in key splicing signals in primary transcripts have been found in many human diseases. The issue of RNA processing is further complicated by differential splicing and numerous alternative splicing events have been linked to fundamental cellular processes and diseases. Now the completion of the human genome project marks the era of functional genomics. Strikingly, up to 60% human genes are found to give rise to multiple mRNA isoforms, and therefore, the regulation of RNA processing may underlie many key control mechanisms in gene expression that are yet to be discovered. Our studies focus on a family of splicing factors called SR proteins, which are not only essential for constitutive splicing but also affect alternative splicing in a dosage dependent manner. Thus, SR proteins may play a key role in the regulation of splicing in humans. Despite extensive biochemical studies on SR proteins, however, their biological functions are largely unexplored. In this proposal, we aim to address some of the fundamental issues concerning SR proteins as potential splicing regulators in vivo: First, we will use conditional knockout mice created by the Cre-LoxP strategy to compare the functional requirement for two prototypical SR proteins SC35 and ASF/SF2 in both normal and pathological processes. Our ultimate goal is to knockout all SR proteins to systematically address the function of SR proteins in biology in the future to test the hypothesis that each SR protein may be specifically required for processing of a subset of pre-mRNAs in vivo and therefore responsible for a unique spectrum of phenotypes in development. Second, we propose to construct an inducible system using mouse embryo fibroblasts derived from conditional knockout mice. This system will allow us to apply biochemical and genetic approaches to conduct functional studies of SR proteins. Finally, we propose to use a number of functional genomic approaches to identify in vivo targets for specific SR proteins. Identification of key targets will help us understand the knockout phenotypes and provide model systems for biochemical studies in order to tie phenotypic studies back to the molecular mechanisms.